DNA amplification fingerprinting (DAF) is one of the recent amplification based nucleic acid scanning technique. This technique developed by Caetano-Anolles et al (1991). DAF utilizes primers of 5 to 12 nucleotide lengths. DAF uses low stringency amplification conditions so that primers can anneal arbitrarily at multiple sites on each template DNA strand and initiate DNA synthesis. DAF method utilizes few reagents in few reaction steps.

Method

 

 A DAF protocol usually involves two major steps. DNA amplification and the separation and visualization of amplification products

Materials for PCR

 

Requirements:

 

PCR, dNTP mix, Taq enzyme, assay buffer, MgCl₂, primer, template DNA, Sterile water, PCR tubes and microcentrifuge tubes (autoclaved) 

Water: Sterile deionized or distilled water should be used for preparing all the reagents and reaction mixes.

Reaction buffers: the manufacturer supplies assay buffer for Taq DNA polymerase.

Magnesium chloride (MgCl₂ ): 25mM stock and at –20^oC

Deoxynucleoside triphosphates (dNTPs): 2.5 mM each of dCTP, dATP, dTTP, dGTP. Readymade of dNTPS are available from many manufacturers. These should be stored at –20˚C

Primers: 200‚µM primers stock store at -20^oc

Enzyme:  Taq DNA polymerase store at –20^oC

Template DNA: Stock DNA should be stored in –20^oC. ( Coconut DNA)

DNA amplification

 

• Amplification mixture is prepared in clean table or in laminar flow. In the following order the reaction mix is added

  Sterile water  3.34‚µl   Assay buffer 1‚µl (10X)   Magnesium chloride       1‚µl (2.5mM)   Deoxynuclioside triphosphates 2‚µl (500‚µm)   Primer      1‚µl (15‚µM)   Taq enzyme 0.66‚µl (2U)   Template coconut DNA 1‚µl (20ng/‚µl)

• While preparing reaction mix with reagents that are common to avoid pipetting errors and aliquots the mix into PCR tube

• amplify the DNA in the thermocycler for 35 cycles using following PCR conditions

  First step  94 ^oC for 3 min   Second step 94^oC for 5 seconds   Third step   55 ^oC for 20 seconds   Forth step 72 ^oC for 30 seconds   Fifth step    go to second step for 34  times   Sixth step  72^oC  for 5 minutes   Seventh step 4^oC forever

• Dilute amplified sample 2 times and denture at 90^oC for 3 minutes before loading into gel and also load 100 base pair molecular markers.

Gel electrophoresis

 

DNA amplification products are separated in a vertical electrophoresis system using 5% non-denaturing polyacrylamide gel of 0.5mm thickness. The DNA fragments are separate according to their molecular weight.

Gels Preparation

 

• Prepare the 40% stock 19:1 acryl amide bis-acrylamide solution store it in dark bottles at 4^o C

• Prepare the 5% working solution containing 7.5 M urea, 40% acryl amide bis-acrylamide, TBE buffer 10X cover the bottle with aluminum foil and store at 4^oC and use before one month.

• Assemble the thoroughly cleaned glass plates in gel casting unit using 0.5mm thick spacers

• Prepare the gel solution by adding 14ml of 5% working solution, 60 ‚µL of 10% (w/v) ammonium per sulfate and 14 ‚µl of TEMED. Mix thoroughly.

• Assemble electrophoresis unit by adding 0.5X TBE buffer to upper tank and lower tank.

• Add 4‚µL of the loading buffer to 8 ‚µL of the final, amplified reaction mix.

• Load this sample into the gel and conduct electrophoresis at 18W for 55 minutes.

• Stop the electrophoresis when the upper dye migrates to the bottom of the gel.

Silver Staining for DNA visualization

 

• Gently place the gel in 10% (v/v) glacial acetic acid for 30 min at room temp.

• Rinse the gel in deionized water twice for about 2 min each.

• Immerse the gel in silver staining solution (250 mg silver nitrate and 375 ‚µl formaldehyde in 250 ml water) for 20 min.

• Pour out the silver stain solution and wash the gel quickly with deionized water with in 10 seconds.

• Immerse the gel in an ice-cold developer solution (10^oC) [7.5 g sodium carbonate, 375 ‚µl formaldehyde, and 50‚µl sodium thiosulfate (10mg in 1ml water) in 250 ml water] until optimal image intensity is obtained. Stop the developing process by immersing the gel in 7.5% ice-cold glacial acetic acid.

• Transfer gel onto the What Mann paper.

• Air-dry the gel or dry using gel drier 70^oC for 30 minutes.

• Gel as such can be preserve as record.

Gel Interpretation

 

• Scoring can be done by presence and absence of band. Bands are sized and matched directly on gels, auto radiographic or photographic films, or photocopies on transparency overlays. 

• The data set generated following band scoring and analysis of fingerprints can be analyzed using computer software NTSYS.

Applications

Protocol can be applicable to assess genetic diversity, the identification of genotype, and segregation and linkage analysis of molecular markers.